Method of quantifying nucleic acid and kit for quantifying nucleic acid

ABSTRACT

It is intended to provide a method of quantifying a nucleic acid and a kit for quantifying a nucleic acid. More specifically, it is intended to provide a standard comprising a synthetic polynucleotide obtained by chemical synthesis which is to be used for forming a calibration curve employed for quantifying a specific target nucleic acid in a sample, a quantification method and a quantification kit using the same, a method of diagnosing a specific disease, etc. The standard is a synthetic polynucleotide obtained by chemical synthesis. Compared with the existing standards comprising biosynthesized polynucleotides, it has an advantage that a target sequence can be precisely obtained by a convenient method. Moreover, the above-described standard suffers from little biological contamination. Therefore, it is highly safe to the environment and factors disturbing highly precise quantification can be lessened therein.

BACKGROUND ART

The present invention relates to a standard preparation that is used todraw up a calibration curve used to quantify a specific target nucleicacid in a sample, a method for quantifying a nucleic acid thereby, or aquantitative kit. The present invention also relates to a method fordiagnosing a specific disease by using the nucleic acid quantitativemethod or quantitative kit, or a medicine comprising the specified geneDNA or the gene product.

Since it is possible to detect a small amount of a target nucleic acidafter amplifying it exponentially by using the PCR (polymerase chainreaction) method, the PCR method is largely used in the fields ofexpression analysis of genes, diagnosis of disease, detection of foodcontaining recombinant genes or determining the presence of recombinantgenes in natural foods. The PCR method is, for example, disclosed inU.S. Pat. No. 4,683,195, U.S. Pat. No. 4,683,202 and U.S. Pat. No.4,965,188 and others in detail. The general PCR method can easily detecta small amount of a target nucleic acid by reacting the sample that maycontain the target nucleic acid with an amplification reagent comprisinga pair of oligonucleotide primer corresponding to the target nucleicacid, reaction substrate (deoxynucleotide triphosphate), and DNApolymerase and the like, and exponentially amplifying the target nucleicacid.

Additionally, the TaqMan method is known, which uses a fluorescent probeand the like in PCR method as a method for quantifying a specific mRNAwith high accuracy. For example, Patent Kokai 2001-204483 discloses aTaqMan method to quantify hTERT mRNA.

Further, a technique using an internal standard has been developed toimprove the accuracy in quantifying a small amount of a target nucleicacid, for example, as disclosed by the publication of Patent KokaiH11-123095. This publication discloses a method for quantifying a targetnucleic acid in which a plasmid including a DNA sequence that behaves asan internal standard is used with an amplification reagent, and the cRNAproduced by this plasmid is used as an internal standard.

By the above method, it is possible to quantify a target nucleic acidmore accurately than with a method that does not use an internalstandard. However, since the RNA as an internal standard is obtainedfrom a plasmid in a reactor by this method, a specific and constantamount of RNA cannot always be prepared depending on the storagecondition of the reagent, the reaction condition, and other factors.Furthermore, a standard preparation produced by the conventional enzymeor organism is complex and is often difficult to prepare. In order toprepare the standard preparation by the conventional method, it isnecessary to extract genome DNA or RNA or mRNA from cells, organs orvirus of the desired species on the basis of the sequence informationand to enzymatically or biologically synthesize the segment of thedesired gene or DNA or RNA having those characteristics. Moreover, thespecies used as materials include some that are difficult to obtain orhave strong pathogens. Furthermore, in the preparation of the standardpreparation, enzymes extracted from organisms or the organisms per seare used, which lead to introduction of biological contaminants thatprevent highly accurate quantification.

Therefore, if there were provided a standard preparation that does nothave the above disadvantages, i.e., a standard preparation obtainable bya convenient method, and having no biological contaminant, or a methodcapable of quantifying a target nucleic acid with a high accuracy usingthe standard preparation, then it would be useful in the fields ofidentification of disease genes, diagnosis of specific diseases,treatment of diseases, or the like.

DISCLOSURE OF THE INVENTION

Therefore, the present invention provides a standard preparation, amethod for quantifying the nucleic acid, a kit for quantifying nucleicacid, a method for diagnosis of a specific diseases, and the like, asdescribed below.

-   (1) A standard preparation useful to quantify or detect a specific    target nucleic acid in a sample, comprising a synthetic    polynucleotide obtained by chemical synthesis.-   (2) The standard preparation of (1), wherein the synthetic    polynucleotide is RNA, DNA or a modification thereof.-   (3) The standard preparation of (1), wherein the synthetic    polynucleotide is RNA or DNA.-   (4) The standard preparation of (3), which is a sense strand if the    synthetic polynucleotide is RNA, or which is an antisense strand if    the synthetic polynucleotide is DNA.-   (5) The standard preparation of (1), wherein the synthetic    polynucleotide is a synthesized part of the target nucleic acid, and    the number of nucleotides is between 60 and 200.-   (6) A kit for quantifying nucleic acid comprising the standard    preparation of any one of claims (1) to (5).-   (7) A kit for quantifying nucleic acid comprising the standard    preparation of any one of claims (1) to (5) and at least one pair of    primers.-   (8) The kit of (7), additionally comprising a fluorescent probe or a    phosphorylated probe.-   (9) The kit of (8), additionally comprising a DNA polymerase.-   (10) The kit of (9), additionally comprising a reverse transferase.-   (11) A kit for quantifying nucleic acid to quantify multiple target    nucleic acids, wherein an amplification reagent comprising a pair of    primers corresponding to the target nucleic acid is loaded with at    each reaction site of a reactor having multiple reaction sites, and    an amplification reagent comprising the standard preparation of any    one of claims 1 to 5 and a pair of primers corresponding to the    standard preparation is loaded at a reaction site which is not    loaded with a pair of primers corresponding to the target nucleic    acid.-   (12) The kit for quantifying nucleic acid of (11), which is used to    diagnose the specific disease, and wherein the multiple target    nucleic acids are DNA or mRNA related to the specific disease.-   (13) The kit for quantifying nucleic acid of (11), which is used to    detect recombinant DNA in food, and wherein the multiple target    nucleic acids are recombinant DNA contained in genetically-modified    food.-   (14) A method for quantifying a specific target nucleic acid in a    sample, which comprises adding an amplification reagents comprising    at least one pair of primers corresponding to a target nucleic acid    to the sample, adding to a chemically synthesized polynucleotide as    a standard preparation, an amplification reagent comprising a pair    of primers corresponding to the synthesized polynucleotide, carrying    out each amplification reaction, measuring the amounts of the    amplified standard preparation and the amplified target nucleic    acid, and calculating the amount of the target nucleic acid before    amplification according to the information obtained by the    measurements.-   (15) The method of (14), wherein the synthetic polynucleotide is    RNA, DNA or a modification thereof.-   (16) The method of (14), wherein the synthetic polynucleotide is    RNA.-   (17) The method of (16), wherein the synthetic polynucleotide is a    sense strand.-   (18) The method of any one of (14) to (17), wherein the synthetic    polynucleotide is a synthesized part of the target nucleic acid, and    the number of nucleotides is between 60 and 200.-   (19) The method of any one of (14) to (18), wherein the sample is an    mRNA sample of human or other animal origins.-   (20) The method of (19), wherein the amplification reagent    additionally comprises a fluorescence probe or a phosphorylated    probe.-   (21) The method of (20), wherein the amplification reagent    additionally comprises a DNA polymerase.-   (22) The method of (21), wherein the amplification reagent    additionally comprises a reverse transferase.-   (23) The method of any one of (20) to (22), wherein (1) a probe    portion of a fluorescence probe or a phosphorylated probe contained    in the amplification reagent comprising at least one pair of primers    corresponding to a target nucleic acid, is a probe consisting of the    nucleic acid of the region between the pair of primers in the target    nucleic acid, and (2) a portion of the a fluorescence probe or a    phosphorylated probe contained in the amplification reagent    comprising the pair of primers corresponding to the synthetic    polynucleotide, is a probe consisting of the nucleic acid of a    region between the pair of primers in the synthetic polynucleotide.-   (24) The method of (23) to measure the amount of the amplified    standard preparation and the amount of the amplified target nucleic    acid using the fluorescence intensity of the fluorescent substance    released from the fluorescence probe or the phosphorylated probe by    DNA polymerase or the amount of phosphate group as an index.-   (25) A method for analyzing SNPs which uses the kit of (11) or the    method of (14).-   (26) A method for diagnosing a specific disease, which uses the kit    of (12) or the method of (14).-   (27) A method for determining if food contains recombinant gene DNA    or not, which uses the kit of (13) or the method of (14).-   (28) A medicine which is specified by the kit of (12) or the method    of (26), which comprises a gene DNA in which expression is    distinctively increased or reduced in a certain cell or tissue, or a    gene product thereof, or an agonist, antagonist, or antibody against    the gene product.

The present invention will be described in detail below.

THE BEST MODE FOR CARRYING OUT THE INVENTION

(Standard Preparation)

The standard preparation of the present invention is used to quantify ordetect a specific target nucleic acid in a sample and is characterizedby comprising a synthetic polynucleotide obtained by chemical synthesis.The “standard preparation” means a standard preparation used to draw upa calibration curve used to calculate the amount of a target nucleicacid before amplification or used to detect a specific nucleic acidsequence such as an SNP. The methods of drawing the calibration curveand calculating the amount of target nucleic acid before amplificationare described later.

The standard preparation used in the present invention is a syntheticpolynucleotide, and is preferably a single strand or double strand RNAobtained by chemical synthesis, a single strand or double strand DNA, ora modification thereof. The “modification” means those having a portionchemically modified, for example: (1) those having a chemically modifiedpurine ring and/or pyrimidine ring (for example, those having methylatedpurine or pyrimidine ring or acylated purine or pyrimidine ring), orthose comprising other heterocyclic ring, (2) those having a chemicallymodified sugar portion(for example, those wherein one or more hydroxylgroups are substituted with halogen or aliphatic groups, or substitutedwith a functional group such as ether or amine), (3) those withbiotinylation, (4) those with FITC conjugation, (5) those modified withdigoxigenin, (6) those with phosphorylation, (7) those modified withperoxidase, (8) those modified with alkaliphosphatase, (9) thosemodified with luciferase.

Since it is possible to obtain entirely by chemical synthesis the neededamount of the desired polynucleotide having a single chemical structure,a chemical synthesized polynucleotide is used in the present invention.

The standard preparation of the present invention is, preferably, asingle strand obtained by chemical synthesis, more preferably, a sensestrand obtained by chemical synthesis. Further, in the case of using asingle chemically synthesized strand, an antisense strand is preferable.Because using a single strand rather than a double strand allows thestandard to more closely resemble the condition of the nucleic acidbeing quantified, the accuracy of the quantification is improved.

The synthesized polynucleotide of the present invention may be asynthesized polynucleotide having the same nucleic acid sequence as thetarget nucleic acid or a portion thereof, and having the similarsequence of the standard preparation obtained by conventionalbiosynthesis. Such standard preparations include, for example, 18SRibosomal RNA, Acidic Ribosomal Protein, β-actin, Cyclophilin,Glyceraldehyde-3-phosphate dehydrogenase, Phosphoglycerokinase,β2-microglobulin, β-Glucuronidase, Hypoxanthine Ribosyl Transferase,Transcription Factor IID/TATA Binding Factor, Transferrin Receptor, andthe like.

It is easy for a skilled artisan to synthesize a polynucleotide for thestandard preparation of the present invention by chemical synthesis onthe basis of the known sequence. Synthesis methods of knownpolynucleotides include, for example, the phosphoamidide method,H-phosphate method, and other known methods.

The phosphoamidide method is disclosed, for example, in Wu, T., Ogilvie,K. K., and Pon, R. T. (1989) “Prevention of chain cleavage in thechemical synthesis of 2′-O-silylated oligoribonucleotides” Nucl. AcidsRes. 17, 3501-3517; Stawinski, J., Stromberg, R., Thelin, M., andWestman, E. (1988) “Studies on the t-butyldimethyl-silyl group as2′-O-protection in oligoribonucleotide synthesis via the H-phosphonateapproach” Nucl. Acids Res. 16, 9285-9288; Scaringe, S A. A., Franklyn,C., and Usman, N. (1990) “Chemical synthesis of biologically activeoligoribonucleotides using b-cyanoethyl protected ribonucleosidephosphoramidites” Nucl. Acids Res. 18, 5433-5441; Chaix, C., Molko, D.and Teoule, R. (1989) “The use of labile base protecting groups inoligoribonucleotide synthesis” Tetrahedron Lett. 30, 71-74; Gasparutto,D., Livache, T., Bazin, H., Duplaa, A. M., Guy, A., Khorlin, A., Molko,D., Roget, A., and Teoule, R. (1992) “Chemical synthesis of abiologically active natural tRNA with its minor bases” Nucleic AcidsRes. 20, 5159-5166; Vinayak, R., Anderson, P. McCollum, C., and Hampel,A. (1992) “Chemical synthesis of RNA using fast oligonucleotidedeprotection chemistry” Tetrahedron Lett. 31, 7269-7272; and the like.Further, H-phosphate method is, for example, disclosed in Garegg, P. J.,Regberg, T., Stawinski, J. and Stromberg, R. (1985). Formation ofinternucleotidic bonds via phosphonate intermediates. Chem. Scripta 25,280-282 ; Garegg, P. J., Regberg, T., Stawinski, J. and Stromberg, R.(1986). Nucleoside hydrogenphosphonates in oligonucleotide synthesis.Chem. Scripta 26, 59-62; Garegg, P. J., Lidh, I., Regberg, T.,Stawinski, J. and Stromberg, R. (1986) ; Nucleoside H-phosphonates. III.Chemical synthesis of oligodeoxyribonucleotides by thehydrogenphosphonate approach. Tetrahedron Lett. 27, 4051-4054; Froehler,B. C., Ng, P. G., and Matteucci, M. D. (1986). Synthesis of DNA viadeoxynucleoside H-phosphonate intermediates. Nucleic Acids. Res. 14,5399-5407; Froehler, B. C., and Matteucci, M. D. (1986). NucleosideH-phosphonates: Valuable intermediates in the synthesis ofoligonucleotides. Tetrahedron Lett. 27, 469-472 and the like. Inaddition, a synthesized polynucleotide used in the present inventionwould preferably be a short one in view of the amplification efficiency,for example, having 60 to 150 mers, more preferably, 60 to 100 mers. Thenumber of the nucleotides of the synthesized polynucleotide in thepresent invention is not specifically limited, and may be in the rangeof 60 to 200, preferably, 60 to 100.

(Nucleic Acid Quantitative Kit)

The kit for quantifying nucleic acid used in the present inventioncomprises a synthesized polynucleotide as the above standardpreparation, and may further comprise at least a pair of primerscorresponding to a target nucleic acid, a probe corresponding to atarget nucleic acid, DNA polymerase, buffer, and the like. These kitsmay comprise, for example, a reagent catalyzing a synthesis of primerextension products, nucleoside triphosphate of a substrate, andimplements used as indicators (for example, if the indicator is biotin,an adipin-enzyme conjugate, the substrate of the enzyme, and chromogen),buffer suitable for the reaction of PCR or hybridization, if necessary.The phrase “pair of primers corresponding to a target nucleic acid” usedin the present invention means a pair of primers consisting of a firstprimer which is complementary or substantially complementary to onestrand of the exon region of a sequence of a target gene (or a genesequence encoding a target mRNA) and a second primer which iscomplementary or substantially complementary to the exon region of thetarget gene sequence on other strand. The exon region between the firstprimer and the second primer is amplified.

In the present invention, a target nucleic acid is amplified using atleast a pair of these primers.

Those skilled in the art can easily design pairs of primers to amplify atarget nucleic acid or pairs of primers to amplify a standardpreparation on the basis of the target nucleic acid (for example, GenomeRes. October 1996; 6 (10): 986-94 reference).

For example, if hMOR1 cDNA is selected as a target nucleic acid, acomplementary sequence to the 1129^(th) to 1210^(th) base of hMOR1 cDNA(SEQ ID NO:1) can be used as a standard preparation by chemicalsynthesis. In this case, 5′-CCTTGGTTACAATCCCAGAAACTAC-3′ (SEQ ID NO: 2)is used as an upstream primer and 5′-AGGCAGCTGTTTGTGTAACCTAGA-3′ (SEQ IDNO:3) as a downstream primer.

It is easy for those skilled in the art to design a probe correspondingto a synthesized polynucleotide used in the present invention or a probecorresponding to a synthesized probe which is a standard preparationaccording to a target sequence (for example, Genome Res. October 1996; 6(10) : 986-94 reference). For example, a probe corresponding to a targetnucleic acid is a probe consisting of a nucleic acid of the regionbetween the first primer and the second primer in the target nucleicacid. A primer corresponding to a synthetic polynucleotide is a probeconsisting of a nucleic acid of the region between the first primer andthe second primer. An adequate oligonucleotide probe used in the presentinvention would preferably have about 15 to about 50 nucleotides, andmore preferably about 25 to about 35 nucleotides. An oligonucleotideprobe can be labeled by incorporating chemical substances and the likethat are detectable by biochemical, immunochemical, or chemical means.Some useful indicators include radioisotopes of P³² or the like;fluorescent substances such as fluorescamine, fluoresceinisothio-cyanate; luminescent substances such as luminal or luciferin;enzymes such as β-galactosidase, peroxidase, alkaliphosphatase; biotin,antibodies and the like. In particular, preferred are fluorescent probeslabeled by fluorescent substances or phosphated probes labeled by P³².

The DNA polymerases used in the present invention are listed asthermostable DNA polymerases having reverse transferase activity and5′→3′ exonuclease activity, for example, Tth DNA polymerase and thelike. In the present invention, known or commercially available buffersmay be used (for example, buffer produced by PEB isosystems; Genome Res.October 1996; 6(10): 986-94 reference ).

In addition, the amount of each reagent that is comprised in thequantitative kit is appropriately determined according to the amount ofa sample or kind of a target nucleic acid.

The more preferable embodiment of the present invention provides a kitfor quantifying nucleic acid to quantify multiple target nucleic acids,wherein each amplification reagent comprising a pair of primerscorresponding to a target nucleic acid is loaded at each of multiplereaction sites (preferably, each reaction site of a reactor havingmultiple reaction sites), and an amplification reagent comprising thestandard preparation of any one of claims 1 to 4 and a pair of primerscorresponding to the standard preparation is loaded at a reaction sitewhich is not loaded with a pair of primers corresponding to the targetnucleic acid. It is possible to detect the presence or absence ofmultiple target nucleic acids or the amount thereof in a sample in asingle procedure by using this quantitative kit.

The multiple reaction sites are not specifically limited as far as thereare two or more reaction sites, generally, 2 to tens of thousands,preferably, 2 to 1,000, more preferably 10 to 800, even more preferably,10 to 300 reaction sites.

In this embodiment, a reactor having multiple reaction sites is used.Each reaction of a sample that may comprise a target nucleic acid witheach amplification reagent, and each of standard preparations consistingof a synthesized nucleotide set up in successive prescribedconcentrations with each amplification reagent are carried out at eachreaction site. The concentration of the standard preparation is notparticularly limited, but preferably is in the range of 10¹ copies to10⁷ copies or more in each container. Each amplification reagentcomprises a pair of primers capable of amplifying a target nucleic acid,or the synthesized polynucleotide, respectively. The reactor used hereinis not particularly limited by the configuration and structure of thereactor as long as there are two or more reaction sites so that a sampleand each amplifying reagent can react in a single procedure. Preferably,the reactors used in the present invention include, for example, a platehaving multiple wells, a reactor having multiple slide glasses, areactor having multiple test tubes and the like. In view of theexperiment space, operability, or the like, preferably, a plate havingmultiple wells can be used. The numbers of the wells of these plates aredetermined by the numbers of the reagents. Preferred are commercial96-well plates and 384-well plates. However, any reactors that have thedesired number of reaction sites according to the numbers of reagentscan be used.

Since the number of amplification reagents is regulated only accordingto the number of target nucleic acids, there is no limit to theirnumber: for example, one could have a quantitative kit comprising astandard preparation consisting of 10 to 800 kinds of amplificationreagents and synthetic oligonucleotides for the targets provided. Inanother embodiment, a kit comprising the standard preparation consistingof 10 to 300 kinds of amplification reagents and a syntheticoligonucleotide for the target is given. If there are many amplificationreagents, it is possible to divide the different sets of amplificationreagents onto multiple plates (for example, on 2 to 10 plates) and carryout quantitative reactions several times.

(Method for Quantifying Nucleic Acids)

The method for quantifying nucleic acids is described below.

According to a method for quantifying a specific target nucleic acid ofthe present invention, amplification reagents comprising at least onepair of primers corresponding to each target nucleic acid are added tothe sample. An amplification reagent comprising a pair of primerscorresponding to the synthetic polynucleotide is added to the chemicalsynthetic polynucleotide as a standard preparation. Amplificationreactions are carried out on both respectively, amounts of the standardpreparation and the amplified target nucleic acid are measured, and theamount of the target nucleic acid before amplification is calculatedaccording to those measurements.

[Sample and Target Nucleic Acid]

First, samples of this invention may comprise a target nucleic acid, andare not particularly limited. The samples of this invention include, forexample, a tissue or mRNA sample derived from cultivated cells of humanor other mammal (e.g. guinea pig, rat, mouse, rabbit, sheep, swine,bovine, feline, canine, monkey, etc.). Examples of such tissues used assamples might include: brain, brain regions (e.g., olfactory bulb,amygdaloid nucleus, cerebral basal bulb, hippocampus, thalamus,hypothalamus, substhanlamic nucleus, cerebral cortex, medulla oblongata,cerebellum, occipital lobe, frontal lobe, temporal lobe, putamen,caudate nucleus, corpus callosum, substantia nigra), spinal cord,hypophysis, stomach, pancreas, kidney, liver, gonad, thyroid,gallbladder, bone marrow, adrenal gland, skin, muscle, lung,gastrointestinal tract (e.g., large intestine and small intestine),blood vessel, heart, thymus, spleen, submandibular gland, peripheralblood, peripheral hemocyte, prostate, testis, ovary, placenta, uterus,bone, joint, skeletal muscle, etc. By quantifying the target mRNAcontained in the mRNA sample, it is possible to analyze the expressionlevel of the target gene at the site where the mRNA is obtained.

Further, use of an mRNA sample derived from a patient having a specificdisease allows easy characterization of a gene related to the disease(for example, GPCR gene). Particularly, in the case of identifying a Gprotein coupled receptor, tyrosine phosphatase receptor, ion channel, orother such genes associated with multiple-gene diseases such as acancer, in which multiple genes seem to be related, the presentinvention is useful to characterize the related gene or protein. This isbecause the expression level of each gene can be measured by onequantitative operation.

By using the synthetic polynucleotide of the present invention asstandard preparation for quantification or detection of specific targetnucleic acid in a sample as described above, it is possible to (1)identify genes of which expression is characteristically increased orreduced in certain cells or tissues by quantitatively analyzing eachexpression level of multiple genes at the same time, and (2) identify acertain gene in a given gene family of which expression ischaracteristically increased or reduced in certain cells or tissues, byanalyzing the expression of multiple genes belonging to that specificgene family at the same time and calculating the absolute value of theexpression level of the gene.

The “multiple genes” are meant to signify two or more genes. There is nospecific upper bound, within limits of feasibility, but the range iscommonly two to several tens of thousands of genes, or preferably, twoto 1,000 genes, or more preferably, 10 to 800 genes, or even morepreferably, 10 to 300 genes.

“The expression level of a gene is characteristically increased orreduced” is meant, compared with the expression of a gene in normalcells or tissues, to refer to a physiologically significant differencein expression, whether small or large. The target nucleic acid family inthe present invention is not specified, but for example, it could beselected from the gene family concerning the G protein coupled receptorgene family, tyrosine phosphatase receptor gene family, ion channel genefamily, or transcription factor, transporter, protein kinase, proteinphosphatase, protease, heat shack protein, ATPase or DNA-binding proteingene family or the like.

The calculation of the level of gene expression or absolute value ofgene expression can be carried out according to the quantitative methodusing a target mRNA as described below.

Also, according to the present invention, use of DNA included inrecombinant gene foods as the target nucleic acid may allow detection ofrecombinant gene foods or presence of recombinant genes in natural foodsobtained without using recombinant gene techniques.

Currently known recombinant gene foods include soybean, potato, corn,tomato, papaya. Further, the recombinant genes in those foods and pairsof primers to detect them, general quantitative methods or the like areknown. For example, they are disclosed in “JAS Analysis Handbookrecombinant gene foods detection/analysis manual quantitative PCR” (H13.April, published by Tokyo IAA Center for Food Quality, Labeling andConsumer Services).

For example, in the detection of corn CB351, a known pair of primersconsists of 5′-CCT TCG CAA GAC CCT TCC TCT ATA-3′ (SEQ ID NO:5) and5′-GTA GCT GTC GGT GTA GTC CTC GT-3′ (SEQ ID NO:6). For the detection ofpapaya 55-1, a known pair of primers consists of 5′-TTA CGG CGA GTT CTGTTA GG-3′ (SEQ ID NO:7) and 5′-CAT GTG CCT GAG AAA TAG GC-3′ (SEQ IDNO:8). For the detection of potato New Leaf Y, a known pair of primersconsists of 5′-AAA AGA GCT GTC CTG ACA GC-3′ (SEQ ID NO:9) and 5′-TCCTCC TGC ATC AAT TGT GT-3′ (SEQ ID NO:10).

In another embodiment of the present invention, the target nucleic acidmay be a G protein coupled receptor, tyrosine phosphatase receptor orion channel coding gene DNA, or mRNA thereof. In this case, by reactingeach amplification reagent comprising a pair of primers corresponding tothe mRNA of a gene belonging to a family such as target G proteincoupled receptor, tyrosine phosphatase receptor, ion channel receptor orthe like with an mRNA sample at each reaction site of the reactor,carrying out amplification reactions, and quantifying mRNA amplifiedproducts, it is possible to measure the expression level of a genebelonging to gene families such as the G protein coupled receptor, thetyrosine phosphatase receptor, the ion channel receptor gene family, orthe like.

In another embodiment of the present invention, by completely preparingall pairs of primers corresponding to the mRNA of a gene belonging togiven gene family such as all of the known G protein coupled receptor,tyrosine phosphatase receptor, ion channel receptor gene families, andthe like, and reacting amplification reagents comprising those pairs ofprimers, respectively at each reaction site, it is possible to measurethe degree of production of each mRNA of the gene belonging to genefamilies such as the G protein coupled receptor, tyrosine phosphatasereceptor, ion channel receptor gene families, and the like in an mRNAsample at the same time. Of course, it is also possible to carry outquantitative reactions several times by appropriately dividing thedifferent sets of amplification reagents onto multiple plates whennecessary.

In another embodiment of the present invention, by using a group ofpairs of primers corresponding to the mRNA of a gene belonging to afamily such as the G protein coupled receptor, tyrosine phosphatase, orthe ion channel receptor gene family, with one quantitativemanipulation, it is possible to characterize the gene belonging to genefamilies such as the G protein coupled receptor, tyrosine phosphatasereceptor, or ion channel receptor gene family highly expressed in thegenes belonging to the gene families that include the group of G proteincoupled receptors, tyrosine phosphatase receptors, ion channelreceptors, and the like.

In addition, the following are currently known as G protein coupledreceptors:

-   (1) Acetylcholine receptors: M₁; M₂; M₃; M₄; M₅-   (2) Adenosine receptors: A₁; A_(2A); A_(2B); A₃-   (3) Adrenoceptors: α1A; α1B; α1D; α2A; α2B; α2C; β1; β2; β3-   (4) Angiotensin receptors: AT1; AT2-   (5) Bombesin receptors: BB1; BB2; bb3-   (6) Bradykinin receptors: B₁; B₂-   (7) Calcitonin, Ainilin, CGRP, and Adrenomedullin receptors:-   (8) Cannabinoid receptors: CB1;CB2-   (9) Chemokine receptors: CCR1; CCR2; CCR3; CCR4; CCR5; CCR6; CCR7;    CCR8; CCR9; CCR10; CXCR1; CXCR2; CXCR3; CXCR4; CXCR5; CX₃CR1; XCR1;-   (10) Cheniotactic receptors: C3_(a); C5_(a); fMLP-   (11) Cholecystokinin and Gastrin receptors: CCK₁; CCK₂-   (12) Corticotropin-releasing factor receptors: CRF₁; CRF₂-   (13) Dopamine receptors: D1; D2; D3; D4; D5-   (14) Endothelin-receptors: ET_(A); ET_(B)-   (15) Galanin receptors: GAL 1; GAL2; GAL3-   (16) Glutamate receptors: mgl₁; mgl₂; mgl₃; mgl₄; mgl₅; mgl₆; mgl₇;    mgl₈-   (17) Glycoprotein hormone receptors: FSH; LSH; TSH-   (18) Histamine receptors: H₁; H₂; H₃; H₄-   (19) 5-HT receptors: 5-HT_(1A); 5-HT_(1B); 5-HT_(1D); 5-ht_(1B);    5-ht_(1F); 5-HT_(2A); 5-HT_(2F); 5-HT_(2C); 5-HT₃; 5-HT₄; 5-ht_(5A);    5-ht_(5B); 5-HT₆; 5-HT₇-   (20) Leukotriene receptors: BLT; CysLT₁; CysLT₂-   (21) Lysophospholipid receptors: edg1; edg2; edg3; edg4-   (22) Melanocorlin receptors: MC₁; MC₂; MC₃; MC₄; MC₅-   (23) Melatonin receptors: MT₁; MT₂; MT₃-   (24) Neuropeptide Y receptors: Y₁; Y₂;Y₄; Y₅; Y₆-   (25) Neurotension receptors: NTS1; NTS2-   (26) Opioids: DOP; KOP; MOP; NOP-   (27) P2Y receptors: P2Y₁; P2Y₂; P2Y₄; P2Y₆; P2Y₁₁; P2Y₁₂-   (28) Peroxisome proliferators: PPAR-α; PPAR-β; PPAR-γ-   (29) Prostanoid receptors: DP; FP; IP; TP; EP₁; EP₂; EP₃; EP₄-   (30) Protease-activated receptors: PAR1; PAR2; PAR3; PAR4-   (31) Somatostatin receptors: sst₁; sst₂; sst₃; sst₄; sst₅-   (32) Tachykinin receptors: NK₁; NK₂; NK₃-   (33) Thyrotropin-releasing hormone receptors: TRH₁; TRH₂-   (34) Urotensin-II receptor:-   (35) Vasoactivate intestinal peptide or pituitary adenylate cyclase    activating peptide receptors: VPAC₁; VPAC₂; PAC₁-   (36) Vasopressin or Oxytocin receptors: V_(1a); V_(1b); V₂; OT

Additionally, the following are known as genes belonging to familiessuch as tyrosine phosphatase receptor, ion channel receptor, or thelike.

-   (37) Ion channel: Na⁺ channels (type I; type II/type IIA; type III;    SCL/NaG; PN1; NaCh6; NaDRG; SkM1/μ1, or SkM2), K⁺ channels (kv; EAG;    KQT; IRK; ROMK; GIRK; K_(ATP) or the like ), Ca²⁺ channels (α1G;    α1E; α1S; α1C; α1D; α1B; α1A; IP3; ryanodine receptor or the like ),    Cl⁻ channels (GABA_(A); GABA_(C); glycine receptors; C1C0; C1C1;    CFTR or the like), non-selective cation channels (nAChR; 5-HT₃;    NMDA; AMPA; P_(2X)ATP; CNG, or the like) or the like.-   (38) Tyrosine phosphatase receptors: insulin receptor; EGF receptor;    or the like.

The nucleic acid quantitative method used in the present invention iscapable of using several kinds of applications other than functionanalysis of human GPCR, SNP analysis, or determination of recombinantgene foods. For example, it is possible to diagnose specific diseases byusing sets of multiple amplification reagents comprising a pair ofprimers detecting mRNA produced by the known disease gene. Because thepresent invention is capable of accurately measuring the expressionlevel of each gene, it has an advantage in providing diagnoses moreaccurately than the known method.

[Amplification of Nucleic Acids]

In the quantitative method of the present invention, a target nucleicacid that may be contained in a sample is amplified by using amplifyingreagents consisting of a pair of primers corresponding to the targetnucleic acid. In the preferred embodiment of the present invention,amplification of a target nucleic acid may be carried out by the knownpolymerase chain reaction (PCR) (see U.S. Pat. No. 4,683,195; U.S. Pat.No. 4,683,202; U.S. Pat. No. 1,965,188 and the others).

When the target is an mRNA, the amplification of the target mRNA may becarried out, for example, by first using viral reverse transcriptase toobtain a cDNA by reverse transcription of the target mRNA, and thenamplifying the obtained cDNA. In a more preferred embodiment of thepresent invention, the amplification of mRNA is carried out by usingreverse transferase-polymerase chain reaction (RT-PCR) (U.S. Pat. No.5,310,652; U.S. Pat. No. 5,322,770; U.S. Pat. No. 5,561,058; U.S. Pat.No.5,641,864; U.S. Pat. No.5,693,517)

In the present invention, many mRNA amplification methods can be used inaddition to the said polymerase chain reaction. These otheramplification methods include, for example, the chain substitution assaymethod (U.S. Pat. No. 5,455,166 and other, reference), the transcriptionamplifying system (TAS) (see U.S. Pat. No. 5,437,990; U.S. Pat. No.5,409,818; U.S. Pat. No. 5,399,491 and others), and the self-sustainedsequence replication system (3SR) (W092/08800 and others, seereferences), among others.

One skilled in the art would easily set the conditions of theseamplifying reactions by varying the kinds of reagents used.

[Quantification of Target Nucleic Acid]

In the next step of the quantification method of the present invention,the amount of amplified nucleic acid product created in the previousstep is quantified. The quantification of this amplified product ispreferably carried out by a method using a probe. According to thepreferred embodiment, the above method should be one using a probelabeled with fluorescent substance.

According to the more preferred embodiment of the present invention, thequantification of target mRNA is carried out by the “TaqMan method” or“5′ nuclease assay method”. (see Proc. Natl. Acad. Sci. USA, vol.88,p7276-7280(1991); U.S. Pat. No. 5,210,015; U.S. Pat. No. 5,487,972; U.S.Pat. No. 5,804,375; U.S. Pat. No.5,804,375 and others). However, ifnecessary, the SYBER Green method, or some hybridization method may beused. In the TaqMan assay method, a probe labeled at the 5′ terminus isused. Also, this probe may be modified at the 3′ terminus to prevent theprobe from working as a primer to synthesize DNA. Examples of thismodification could include the addition of a phosphate group or somefluorescent substance to the 3′ terminus. Amplification of the targetmRNA may be carried out by using a DNA polymerase having 5′→3′exonuclease activity, for example, Tth DNA polymerase. Any probes thathybridize downstream from the abovementioned primer on the target mRNAare removed by the 5′→3′ exonuclease activity of DNA polymerase duringthe amplification reaction. During each new amplification step of thetarget region, the probe is removed and the labeled substance (forexample, phosphate group or fluorescent substance), with which the probewas modified, is released. By quantifying the amount of this releasedlabeling substance, the amount of target mRNA produced can be indirectlymeasured.

Known methods are used to detect the released target substancequantitatively. In the preferred method, the said probe is labeled atthe 5′ and 3′ terminus by two fluorescent substances, and each substancehas the ability to suppress the fluorescence of the other substance.While this probe is hybridized to template DNA, the fluorescence emittedby the two substances is suppressed by their reciprocal activity uponeach other. However, when the probe is removed by the 5′-3′ exonucleaseactivity of the DNA polymerase, these substances will begin tofluoresce. This fluorescence is increased according to progress of theamplification reaction, and this increase in fluorescence is monitored.

[Drawing up the Calibration Curve and Calculating the Amount of TargetNucleic Acid Before Amplification]

The present invention provides prescribed amount of a synthesizedpolynucleotide as a standard preparation. Thus, a sample comprising atarget nucleic acid can be quantified based on the “calibration curve”drawn up after amplification of the standard preparation.

A way of drawing up this calibration curve is disclosed, for example, inthe publication of Japanese Patent No. H11-123095. To draw up thecalibration curve, the amount of polynucleotide as the standardpreparation produced in polymerase chain reaction is plotted as againstvaried known amounts of RNA present before amplification. In order toensure a high level of accuracy, the calibration curve is drawn up bycarrying out the amplification reaction using a series of dilutions thatgradually change the concentration of the amplification reactionmixture. This calibration curve is drawn up by plotting the amount ofinternal standard preparation or the target nucleic acid amplified overa given number of amplification cycles.

The amount of the target nucleic acid before amplification is determinedby comparing the amount of the amplified target nucleic acid with theabove calibration curve. The standard preparation and a set of dilutionseries of target nucleic acid is amplified at another reaction siteunder the same conditions; the reaction is stopped at the exponentialstage of amplification; and the amount of target nucleic acid present inthis sample before the amplification is determined to be extrapolatedaccording to the calibration curve drawn up by using the standardpreparation.

[Method for Diagnosing a Disease and Medicine for Treating the Disease]

According to the above method (23) of the present invention, geneexpression analysis may be carried out using mRNA sample derived from apatient. This expression analysis provides information that can be usedto diagnose the patient by detecting the characteristic expression of aspecific disease-related gene. In the present invention, by analyzingthe gene expression of a specific disease-related gene family(particularly, the disease-related GPCR gene family of which genes arefound in some patients), one can determine which gene among many isresponsible for the characteristic gene expression in a singleoperation.

Therefore, a medicine comprising an agonist, antagonist, antibodyagainst the gene product of the specified gene, or a DNA coding for thegene product may be particularly effective for patients that are thesubject of such a diagnosis. The present invention makes it possible tospecify plural abnormally expressed genes and even to quantify withaccuracy the expression level of those genes. Therefore, it is possibleto select plural agonists, antagonists, or antibodies as an adequateprescription for the patient and to adjust the amount of thisprescription according to the patient's expression level of thedisease-related gene. That is, the present invention allows preparationof a tailor-made medicine that will be prescribed specifically for thepatient.

More specifically, for example, the following treatments may beeffective in a patient who cannot expect a physiological function of aligand to a certain receptor protein because of reduced levels of thereceptor protein (as in the case of a patient with a deficiency syndromeof the receptor protein). It is possible to increase the amount of thisreceptor protein in the patient to ensure sufficient activities of theligand by {circle over (1)} administering said receptor protein to thepatient by prescribing a sufficient amount of the receptor protein,{circle over (2)} either (i) administering a DNA of the presentinvention coding for the needed receptor protein to the patient, andexpressing the DNA, or (ii) implanting a cell within the patient afterinserting a DNA coding for the receptor protein into the desired celland expressing the DNA.

The medicine of the present invention is effective in the preservationor treatment of a disease related to a specified gene, for example,diseases of the central nervous (for example, Alzheimers disease,dementia, eating disorders, or the like), endocrinopathy (for example,hypertension, abnormal gonadal function, abnormal thyroid function,abnormal pituitary function, or the like), metabolic disease (forexample, diabetes, lipidosis, hyperlipidemia or the like), cancer (forexample, non-parvicellular lung cancer, ovarian cancer, prostate cancer,stomach cancer, urinary bladder cancer, breast cancer, cervical cancer,colon cancer, rectal cancer, or the like).

When a gene product of a gene specified by the present invention (forexample, receptor protein), an agonist, antagonist or antibody thereto,or a DNA encoding the gene, is used as a prophylactic/therapeutic agentas mentioned above, a pharmaceutical preparation can be prepared in aconventional manner.

On the other hand, where the DNA encoding the protein of the presentinvention (hereinafter sometimes referred to as the DNA of the presentinvention) is used as a prophylactic/therapeutic agent as describedabove, the DNA itself is administered; alternatively, the DNA isinserted into an appropriate vector such as a retrovirus vector,adenovirus vector, adenovirus-associated virus vector, etc. and thenadministered in a conventional manner. The DNA of the present inventionmay also be administered as naked DNA, or with adjuvants to assist itsuptake by gene gun or through a catheter such as a catheter with ahydrogel.

For example, {circle over (1)} the medicine of the present invention canbe used orally, for example, in the form of tablets which may be sugarcoated if necessary, capsules, elixirs, microcapsules etc., orparenterally in the form of injectable preparations such as a sterilesolution and a suspension in water or with another pharmaceuticallyacceptable liquid. These preparations can be manufactured by mixing{circle over (1)} the protein of the present invention with aphysiologically acceptable known carrier, a flavoring agent, anexcipient, a vehicle, an antiseptic agent, a stabilizer, a binder, etc.in a unit dosage form required in a generally accepted manner that isapplied to making pharmaceutical preparations. The effective componentin the preparation is controlled in such a dose that an appropriate doseis obtained within the specified range given.

Additives miscible with tablets, capsules, etc. include a binder such asgelatin, corn starch, tragacanth and gum arabic, an excipient such ascrystalline cellulose, a swelling agent such as corn starch, gelatin andalginic acid, a lubricant such as magnesium stearate, a sweetening agentsuch as sucrose, lactose and saccharin, and a flavoring agent such aspeppermint, Gaultheria adenothrix oil, and cherry. When the unit dosageis in the form of capsules, liquid carriers such as oils and fats mayfurther be used together with the additives described above. A sterilecomposition for injection may be formulated by conventional proceduresused to make pharmaceutical compositions, e.g., by dissolving orsuspending the active ingredients in a vehicle such as water forinjection with a naturally occurring vegetable oil such as sesame oiland coconut oil, etc. to prepare the pharmaceutical composition.Examples of an aqueous medium for injection include physiological salineand an isotonic solution containing glucose and other auxiliary agents(e.g., D-sorbitol, D-mannitol, sodium chloride, etc.) and may be used incombination with an appropriate dissolution aid such as an alcohol(e.g., ethanol or the like), a polyalcohol (e.g., propylene glycol andpolyethylene glycol), a nonionic surfactant (e.g., polysorbate 80™ andHCO-50), etc. Examples of the oily medium include sesame oil and soybeanoil, which may also be used in combination with a dissolution aid suchas benzyl benzoate and benzyl alcohol.

The prophylactic/therapeutic agent described above may further beformulated with a buffer (e.g., phosphate buffer, sodium acetate buffer,etc.), a soothing agent (e.g., benzalkonium chloride, procainehydrochloride, etc.), a stabilizer (e.g., human serum albumin,polyethylene glycol, etc.), a preservative (e.g., benzyl alcohol,phenol, etc.), an antioxidant, etc. The thus-prepared liquid forinjection is normally filled in an appropriate ampoule.

Since the thus obtained pharmaceutical preparation is safe and lowtoxic, the preparation can be administered to humans or mammals (e.g.,rats, rabbits, sheep, swine, bovine, cats, dogs, monkeys, etc.).

The dose of the medicine of the present invention varies depending onthe subject to which it will be administered, target organs, conditions,routes for administration, etc.; in oral administration, e.g., an adultpatient, the dose is normally about 0.1 mg to about 100 mg, preferablyabout 1.0 to about 50 mg, and more preferably about 1.0 to about 20 mgper day (for 60 kg body weight). In parenteral administration, thesingle dose varies depending on the subject to which it will beadministered, target organ, conditions, routes for administration, etc.,but it is desirable, e.g., for an adult patient, to administer theactive ingredient intravenously in a daily dose of about 0.01 to about30 mg, preferably about 0.1 to about 20 mg, and more preferably about0.1 to about 10 mg (for 60 kg body weight). For other animal species,the corresponding dose as converted per 60 kg body weight can beadministered.

(Denotation of Abbreviations)

In the specification and drawings, the codes of bases, amino acids,compound and others are denoted in accordance with the IUPAC-IUBCommission on Biochemical Nomenclature or by the common codes in theart, examples of which are shown below. For amino acids that may havethe optical isomer, L form is presented unless otherwise indicated.

DNA: Deoxyribonucleic acid

cDNA: Complementary deoxyribonucleic acid

A: Adenine

T: Thymine

G: Guanine

C: Cytosine

RNA: Ribonucleic acid

mRNA: Messenger ribonucleic acid

dATP: Deoxyadenosine triphosphate

dTTP: Deoxythymidine triphosphate

dGTP: Deoxyguanosine triphosphate

dCTP: Deoxycytidine triphosphate

ATP: Adenosine triphosphate

EDTA: Ethylenediamine tetraacetic acid

SDS: Sodium dodecyl sulfate

Gly: Glycine

Ala: Alanine

Val: Valine

Leu: Leucine

Ile: Isoleucine

Ser: Serine

Thr: Threonine

Cys: Cysteine

Met: Methionine

Glu: Glutamic acid

Asp: Aspartic acid

Lys: Lysine

Arg: Arginine

His: Histidine

Phe: Phenylalanine

Tyr: Tyrosine

Trp: Tryptophan

Pro: Proline

Asn: Asparagine

Gln: Glutarnine

pGlu: Pyroglutamic acid

Tos: P-toluenesulfonyl

CHO: Formyl

Bzl: Benzyl

Cl₂Bzl: 2,6-dichlorobenzyl

Bom: Benzyloxymethyl

Z: Benzyloxycarbonyl

Cl-Z: 2-chlorobenzyloxycarbonyl

Br-Z : 2-bromobenzyloxycarbonyl

Boc: T-butoxycarbonyl

DNP: Dinitrophenol

Trt: Trityl

Bum: T-butoxymethyl

Fmoc: N-9-fluorenylmethoxycarbonyl

HOBt: 1-hydroxybenztriazole

HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine

HONB: 1-hydroxy-5-norbornene-2,3-dicarboximide

DCC: N,N′-dicyclohexylcarbodiimide

The sequence identification numbers in the sequence listing of thespecification indicates the following sequence, respectively.

[SEQ ID NO:1]

This shows the base sequence of h MOR1.

[SEQ ID NO:2]

This shows the base sequence of an up stream primer N-917F used inExample 1.

[SEQ ID NO:3]

This shows the base sequence of a down stream primer N-998R used inExample 1.

[SEQ ID NO:4]

This shows the base sequence of probe N-945T used in Example 1.

[SEQ ID NO:5]

This shows the base sequence of primer for detection of corn CB351.

[SEQ ID NO:6]

This shows the base sequence of primer for detection of corn CB351.

[SEQ ID NO:7]

This shows the base sequence of primer for detection of papaya 55-1.

[SEQ ID NO:8]

This shows the base sequence of primer for detection of papaya 55-1.

[SEQ ID NO:9]

This shows the base sequence of primer for detection of potato New LeafY.

[SEQ ID NO:10]

This shows the base sequence of primer for detection of potato New LeafY.

EXAMPLES.

The present invention is described in detail below with reference toEXAMPLES, which are not deemed to limit the scope of the presentinvention.

Example 1

(1) Drawing up the Calibration Curve for Chemically Synthesized DNAEncoding a Partial Sequence of hMOR1

This example describes drawing up the calibration curve for chemicallysynthesized DNA encoding a partial sequence of hMOR1 cDNA (SEQ ID NO:1:GenBank accession number L25119) by TaqMan method.

(2) Samples

The amplification was carried out using serial dilution solution of thechemically synthesized DNA encoding a partial sequence of hMOR1 cDNA(hMOR1T/M). The sequence of hMOR1COM of the present inventioncorresponds to the complementary sequence from the 1129th to the1210^(th) bp of hMOR1 cDNA. After synthesis byβ-cyanoethylphosphoamidaide solid-synthesis method (chemical synthesismethod), ammonia cleavage treatment was carried out, followed bypurification by polyacrylamide modified gel electrophoresis.

(3) Amplification Primers and Detection Probes

The amplification of hMOR1 cDNA partial sequence region was carried outby using up stream primer N-917F having the sequence5′-CCTTGGTTACAATCCCAGAAACTAC-3′ (SEQ ID NO:2), and down stream primerN-998R having the sequence 5′-GCAGCTGTTTGTGTAACCTAGA-3′ as a pair ofprimers.

The above up stream primer, N-917F hybridizes the complementary sequencefrom the 1129th-1153th bp of hMOR1 cDNA (SEQ ID NO:1). The above downstream primer, N-998R hybridizes the sequence from the 1187th-1210^(th)bp of hMOR1 cDNA (SEQ ID NO:1). These primers catalyze the amplificationof an 82 base pair product comprising one part of the entire length ofthe hMOR1 cDNA sequence.

The detection was carried out by using N-945T, having5′-CCAGACTGTITCTTGGCACTTCTGCATTG-3′ (SEQ ID NO:4) as a probe. This probehybridizes to the complementary sequence from the 1157^(th)-1185^(th) bpof hMOR1 cDNA (SEQ ID NO:1).

In order to make it possible to carry out the detection with the TaqManmethod, the above probe was labeled with a fluorescein type fluorescentdye (FAM: reporter) at the 5′ terminal and a rhodamine type fluorescentdye (TAMRA: quencher) at the 3′ terminal.

In the labeled probe, the fluorescence of the reporter is suppressedwhen it does not hybridize, because of a migration phenomenon offluorescent resonant energy. To prevent extension of the above probe byDNA polymerase during amplification, the 3′ terminal of the above probeis blocked with a phosphoric acid.

(4) Amplification

Each PCR amplification was carried out with a total reaction amount of20 μl using TaqMan™ Universal PCR Master MIX (Applied Bio Systems JapanCo. Ltd,.). The following is the final reagent concentration : samplegene, 1X TaqMan™ Universal PCR Master MIX (comprising AmpliTaq Gold™ DNApolymerase, AmpErase™ Urasil-N-glycosylase (UNG), ROX and others), 900nM of each primer, 200 nM of probe.

The amplification reaction was carried out with ABI PRISM (trade mark)7900HT sequence detection system (Applied Bio Systems Japan Co. Ltd.).The following is a profile of the thermal cycle used.

The time and temperature of the thermal cycle and the incubation in AmpErase UNG reaction: two minutes at 50° C., activation of the AmpliTaqGold DNA polymerase: ten minutes at 95° C.,denaturation/annealing/extension: 40 cycles: 15 seconds at 95° C., 1minute at 60° C.

(5) Quantitative TaqMan Analysis

In the TaqMan analysis, during amplification, the probe hybridizing tothe above target sequence is hydrolyzed from the 5′ terminal by 5′-3′exonuclease reaction by the above DNA polymerase. Then, the reporterfluorescent dye is released and the fluorescence intensity increases.

The stored amplified product was quantified by measuring the increase offluorescent intensity of the reporter dye in the reaction solution. Atthe same time, the fluorescent intensity of the fluorescent reference(fluorescent dye: ROX) was also measured to determine experimental errorin the reaction solution. During each amplification cycle, the abovereporter fluorescent dye and reference dye excite at the wavelength oflight near the greatest excitation. Emission of the above reporterfluorescent dye and reference fluorescent dye are measured at the timeof the greatest emission. This frequency is determined by the ABI PRISM™7900HT sequence detection system in advance. If another detectioninstrument is used, then an adequate frequency should be selected.

The value of the fluorescence was analyzed by 7900HT SDS software(Applied Bio Systems Japan Co. Ltd.). First, the fluorescent intensityof the reporter fluorescent dye was standardized by a referencefluorescent dye, and then a standardized reporter signal (Rn) wascalculated. Then, the value calculated as being the average value(baseline) of relatively constant Rn during each cycle of the PCRprimary cycle was designated as Δ Rn. When the amplification curve wasplotted, the cycle number which analysis algorithm detected an increaseof ΔRn to the number of cycles, an increase of fluorescent signal (ΔRn)corresponding to an exponential amplification of the amplificationproduct for the first time was designated as the Threshold Cycle(C_(T)). In particular, in order to determine the C_(T), PCR primarycycles (3-15 cycles) that are not considered to have achievedexponential amplification of the amplification product were used as abaseline, and the standard deviation of an average ΔRn in this cycle wascalculated. Then, the value of this standard deviation was multipliedten times and defined as the “Threshold”. The cycle number correspondingto this Threshold value on each amplification curve was defined asC_(T).

During the period of exponential amplification of the aboveamplification product, the C_(T) is proportional to the logarithm of thefirst target copy number. Accumulation of the amplification product inthe later stage cycles inhibits the reaction and at last leads to theplateau of the reaction.

(6) Results

The following are C_(T) values obtained from each sample. Each C_(T)represents the average obtained from four reactions. Sample C_(T) value10⁷ copies of hMOR1T/M 16.3 10⁶ copies of hMOR1T/M 19.2 10⁵ copies ofhMOR1T/M 22.5 10⁴ copies of hMOR1T/M 26.0 10³ copies of hMOR1T/M 29.210² copies of hMOR1T/M 32.9 0 copy of hMOR1T/M above 40.0

The calibration curve was prepared using the C_(T) value obtained fromthe amplification of a known amount of template hMOR1COM (standardsample). In particular, the calibration curve was drawn up by plottingC_(T) against this known primary amount of standard sample (logarithmvalue). For an approximated curve, the following linear equation wasused.C _(T)=(Log [DNA]_(T)-Log[DNA]₀)/Log(1+e)(wherein [DNA]₀ is a primary concentration, [DNA]_(T) is a concentrationof an amplification product at a C_(T) cycle, e is an averageamplification efficiency, and Log(X) is a logarithm where X represents abase of log 10). The 7900HT SDS software was used to to determineparameters.

By using the value of C_(T) obtained from samples, the followingcalibration curve was obtained:C _(T)=39.34−3.331×Log [DNA]₀

-   -   Correlation coefficient R2=0.999    -   Average amplification efficiency e=99.6%

Example 2

(1) Extraction of RNA and Synthesis of DNA

Prostate cancer cells (LNCaP-FGC cells) were cultured untilpreconfluent. After removing the cells with 0.25% trypsin-lmM EDTA(Invitrogen Co. Ltd.) and counting the number of cells, total RNA wasextracted and purified according to the manual instructions of theRNeasy mini KIT (QUAGEN Co. Ltd.). The first strand cDNA was synthesizedfrom the extracted RNA according to the manual instructions of theSuperScript II (Invitrogen Co. Ltd.), and after ethanol precipitation,the cDNA was eluted and used as below. Synthesized cDNA was dissolved inTE to correspond to 10 mg/ml RNA and diluted to correspond to 5 ng/μl inTE comprising 50 μg/ml yeast tRNA. Five (5) μl of diluted cDNA solution(corresponding to 25 ng of RNA) was used as a measuring sample for aquantification of mRNA of one kind of GPCR.

(2) Quantification of GPCR mRNA Using Chemical Synthesized StandardPreparation

Based upon the known sequences of 160 kinds of GPCRs, primers and probeswere designed using Primer Express™ software (Applied Bio Systems JapanCo. Ltd.). Further, (−) strand DNA having a sequence between the primerswas chemically synthesized. After diluting chemically synthesized DNA to10⁶ copies /5 μl with TW comprising 50 μg/ml yeast tRNA, dilution serieswere prepared until a concentration of 10² copies /5 μl each wereachieved at 10×. The measurement of one kind of GCPR mRNA was carriedout by dispensing 5μ of each of five kinds of dilution series of thosechemically synthesized products and one of the above measuring samplesby duplex. As the amplification reagents, TaqMan™ Universal PCR Masterkit (Applied Bio Systems Japan Co. Ltd.) and the above designed TaqMan™Probe Kit (Applied Bio Systems Japan Co. Ltd.) were used. Afterpreparing an amount of 15 μl solution of the amplification reagents,those solutions were added to each well containing the above standardpreparation or the measuring sample. The final concentration of eachprimer and probe was adjusted according to manual instructions. TaqMan™PCR was carried out by ABI PRISM™ 7900HT sequence detection system(Applied Bio Systems Japan Co. Ltd.), using the thermal cycles describedin the manual of TaqMan™ universal PCR Master Mix (Applied Bio SystemsJapan Co. Ltd.)

The quantitative TaqMan analysis of the amplification product wascarried out by 7900HT SDS software (Applied Bio Systems Japan Co. Ltd.).Using the above conditions, quantitative measurement of the 32 kinds ofGPCR was carried out in one well of a 384-well plate. Use of five platesof a 384-well plate allowed accurate quantification of all theaforementioned 160 GPCRs expressed in the prostate cancer cell strain(LNCaP-FGC cells).

Industrial Applicability

The standard preparation of the present invention is a syntheticpolynucleotide with the advantage that a desired sequence can accuratelybe obtained by a concise method compared to the standard preparationconsisting of a polynucleotide obtained by conventional biosynthesis.Further, the standard preparation of the present invention is notbiologically contaminated. Thus, the standard preparation of the presentinvention is safe for the environment, and has the additional advantageof reducing factors that inhibit highly accurate quantification.

Moreover, when a single strand polynucleotide is used as the standardpreparation, the quantitative method and the quantitative kit of thepresent invention allow more accurate quantification because ofsimilarity of the standard used to substance to be quantified.

Furthermore, the other embodiment of the quantitative method and thequantitative kit of the present invention can, with a singlemanipulation, detect with high-sensitivity an amount of the multipletarget nucleic acids in samples that may contain multiple target nucleicacids. Therefore, the present invention provides a system that can carryout expression analysis of the target nucleic acid withhigh-sensitivity.

Furthermore, the quantification method and quantification kit of thepresent invention make it possible to diagnose specific diseases andexamine genetically modified foods to determine if recombinant DNA ispresent.

1. A standard preparation useful to quantify or detect a specific targetnucleic acid in a sample, comprising a synthetic polynucleotide obtainedby chemical synthesis.
 2. The standard preparation of claim 1, whereinthe synthetic polynucleotide is RNA, DNA or a modification thereof. 3.The standard preparation of claim 1, wherein the syntheticpolynucleotide is RNA or DNA.
 4. The standard preparation of claim 3,which is a sense strand if the synthetic polynucleotide is RNA, or whichis an antisense strand if the synthetic polynucleotide is DNA.
 5. Thestandard preparation of claim 1, wherein the synthetic polynucleotide isa synthesized part of the target nucleic acid, and the number ofnucleotides is between 60 and
 200. 6. A kit for quantifying nucleic acidcomprising the standard preparation of any one of claims 1 to
 5. 7. Akit for quantifying nucleic acid comprising the standard preparation ofany one of claims 1 to 5 and at least one pair of primers.
 8. The kit ofclaim 7, additionally comprising a fluorescence probe or aphosphorylated probe.
 9. The kit of claim 8, additionally comprising aDNA polymerase.
 10. The kit of claim 9, additionally comprising areverse transferase.
 11. A kit for quantifying nucleic acid to quantifymultiple target nucleic acids, wherein an amplification reagentcomprising a pair of primers corresponding to the target nucleic acid isloaded at each reaction site of a reactor having multiple reactionsites, and an amplification reagent comprising the standard preparationof any one of claims 1 to 5 and a pair of primers corresponding to thestandard preparation is loaded at a reaction site which is not loadedwith a pair of primers corresponding to the target nucleic acid.
 12. Thekit for quantifying nucleic acid of claim 11, which is used to diagnosea specific disease, and wherein the multiple target nucleic acids areDNA or mRNA related to the specific disease.
 13. The kit for quantifyingnucleic acid of claim 11, which is used to detect recombinant DNA infood, and wherein the multiple target nucleic acids are recombinant DNAcontained in genetically-modified food.
 14. A method for quantifying aspecific target nucleic acid in a sample, which comprises adding anamplification reagent comprising at least one pair of primerscorresponding to a target nucleic acid to the sample, adding, to achemically synthesized polynucleotide as a standard preparation, anamplification reagent comprising a pair of primers corresponding to thesynthesized polynucleotide, carrying out each amplification reaction,measuring the amounts of the amplified standard preparation and theamplified target nucleic acid, and calculating the amount of the targetnucleic acid before amplification according to the information obtainedby the measurements.
 15. The method of claim 14, wherein the syntheticpolynucleotide is RNA, DNA or a modification thereof.
 16. The method ofclaim 14, wherein the synthetic polynucleotide is RNA.
 17. The method ofclaim 16, wherein the synthetic polynucleotide is a sense strand. 18.The method of any one of claims 14 to 17, wherein the syntheticpolynucleotide is a synthesized part of the target nucleic acid, and thenumber of nucleotides is between 60 and
 200. 19. The method of any oneof claims 14 to 17, wherein the sample is an mRNA sample of human orother animal origins.
 20. The method of claim 19, wherein theamplification reagent additionally comprises a fluorescence probe or aphosphorylated probe.
 21. The method of claim 20, wherein theamplification reagent additionally comprises a DNA polymerase.
 22. Themethod of claim 21, wherein the amplification reagent additionallycomprises a reverse transferase.
 23. The method of any one of claim 20,wherein (1) a probe portion of a fluorescence probe or a phosphorylatedprobe contained in the amplification reagent comprising at least a pairof primers corresponding to a target nucleic acid, is a probe consistingof the nucleic acid region between the pair of primers in the targetnucleic acid, and (2) a probe portion of a fluorescence probe or aphosphorylated probe contained in the amplification reagent comprisingthe pair of primers corresponding to the synthetic polynucleotide, is aprobe consisting of the nucleic acid region between the pair of primersin the synthetic polynucleotide.
 24. The method of claim 23, whichcomprises measuring the amount of the amplified standard preparation andthe amount of the amplified target nucleic acid using the fluorescenceintensity of the fluorescent substance released from the fluorescenceprobe or the phosphorylated probe by DNA polymerase or the amount ofphosphate group as an index.
 25. A method for analyzing SNPs, which usesthe kit of claim 11 or the method of claim
 14. 26. A method fordiagnosing a specific disease, which uses the kit of claim 12 or themethod of claim
 14. 27. A method for determining if food containsrecombinant gene DNA or not, which uses the kit of claim 13 or themethod of claim
 14. 28. A medicine specified by the kit of claim 12 orthe method of claim 26, which comprises a gene DNA in which expressionis distinctively increased or reduced in a certain cell or tissue, agene product thereof, or an agonist, antagonist or antibody against thegene product.
 29. The method of claim 21, wherein (1) a probe portion ofa fluorescence probe or a phosphorylated probe contained in theamplification reagent comprising at least a pair of primerscorresponding to a target nucleic acid, is a probe consisting of thenucleic acid region between the pair of primers in the target nucleicacid, and (2) a probe portion of a fluorescence probe or aphosphorylated probe contained in the amplification reagent comprisingthe pair of primers corresponding to the synthetic polynucleotide, is aprobe consisting of the nucleic acid region between the pair of primersin the synthetic polynucleotide.
 30. The method of claim 29, whichcomprises measuring the amount of the amplified standard preparation andthe amount of the amplified target nucleic acid using the fluorescenceintensity of the fluorescent substance released from the fluorescenceprobe or the phosphorylated probe by DNA polymerase or the amount ofphosphate group as an index.
 31. The method of claim 22, wherein (1) aprobe portion of a fluorescence probe or a phosphorylated probecontained in the amplification reagent comprising at least a pair ofprimers corresponding to a target nucleic acid, is a probe consisting ofthe nucleic acid region between the pair of primers in the targetnucleic acid, and (2) a probe portion of a fluorescence probe or aphosphorylated probe contained in the amplification reagent comprisingthe pair of primers corresponding to the synthetic polynucleotide, is aprobe consisting of the nucleic acid region between the pair of primersin the synthetic polynucleotide.
 32. The method of claim 31, whichcomprises measuring the amount of the amplified standard preparation andthe amount of the amplified target nucleic acid using the fluorescenceintensity of the fluorescent substance released from the fluorescenceprobe or the phosphorylated probe by DNA polymerase or the amount ofphosphate group as an index.